Hiding in plain sight: Cryptic enemies are found on cochineal (Hemiptera: Dactylopiidae), a scale insect of economic and cultural significance

Abstract Cochineal is the common name for cactus‐feeding scale insects in the Dactylopiidae. These ruby‐red insects include the domesticated dye insect Dactylopius coccus. Dactylopius coccus and congeners have been introduced around the world, some accidentally, to become pests of prickly pear cactus species (Opuntia), and some intentionally, for dye production or biological control of pest Opuntia. In the northern Sonoran Desert (Tucson, AZ, USA), we studied the enemy complex of D. opuntiae and D. confusus on Opuntia and characterized two cryptic enemies, a coccinellid beetle predator and a parasitoid wasp. (1) Hyperaspis sp. The coccinellid predator Hyperaspis trifurcata shares a niche with a similar, typically all‐black beetle. Morphological data, crossing tests, and phylogenetic results showed the black beetle to be a distinct, undescribed species in the genus Hyperaspis, with a rare spotted phenotype that is similar in appearance to H. trifurcata. Crossing tests among black and spotted forms showed the spotted morph is inherited as a single‐locus dominant allele. (2) Formicencyrtus thoreauini. Rearing of this ant‐like parasitoid wasp (Encyrtidae) in pure culture of D. opuntiae showed it to be a semi‐gregarious primary parasitoid of cochineal. To our knowledge, this is the first confirmed instance of a cochineal parasitoid. Observations of development show early instar larvae keep their posterior end within the egg chorion, attached to an aeroscopic plate with a connection to the cochineal body wall. Late instar larvae are eventually surrounded by a membrane, likely of larval origin. Wasps then pupate in a dry air‐filled chamber within the desiccated scale remains before chewing out as an adult. Both Hyperaspis sp. and F. thoreauini may have restricted distributions. Hyperaspis sp. does not appear to be a member of the cochineal community in Mexico or Texas, and scant records suggest F. thoreauini may also be restricted to the Southwestern USA.


| INTRODUC TI ON
Specialist enemies that attack cochineal are notable because of the long history of human fascination with their prey. The iconic bright red scale insects, clothed in cottony wax, include 11 species in the monogeneric family Dactylopiidae (García Morales et al., 2016).
Long before European arrival to the New World, the Aztecs domesticated Dactylopius coccus Costa for its red hemolymph, the source of a brilliant red dye (Greenfield, 2006), making this species one of only a few domesticated insects, along with the honeybee and silk moth. In the early 19th century, over 3 million pounds of dried insects were exported from Mexico in a single year (1802), before alternative red dyes and overseas production caused the profitability of the local industry to decline (Hunter et al., 1912). While the prickly pear cactus species in the genus Opuntia and all cochineal species are native to the Americas, both host plants and cochineal have been widely introduced to warm arid climates around the world in the last 200 years, some Dactylopius for cochineal dye production (often without regard to species identification) and some Opuntia for fodder, fruit production, and erosion control.
Opuntia and cochineal introductions have long spurred conflicts of interest since prickly pear is considered a valuable plant in the Middle East and North Africa. For example, Opuntia is a forage and fruit crop in Morocco and a dry-adapted hedgerow species of cultural significance in Israel (Bouharroud et al., 2019;Mendel et al., 2020;Paterson et al., 2021;Paterson & Witt, 2022;Spodek et al., 2013). A recent invasion of Dactylopius opuntiae (Cockerell) in Israel, Lebanon, and Morocco caused significant cactus mortality in plantations and landscapes (Bouharroud et al., 2019;Spodek et al., 2013). In Israel, a biological control program for D. opuntiae was launched and two specialist predators, a coccinellid beetle, Hyperaspis trifurcata Schaeffer, and a chamaemyiid fly, Leucopis bellula Williston, were introduced (Mendel et al., 2020). Conversely, prickly pear species are invasive plant pests of rangeland in several areas, including Kenya, Southern Africa, India, and Australia. In these locations, cochineal species have been important classical biological control agents for the cactus (Annecke & Moran, 1978;Novoa et al., 2019;Paterson et al., 2021;Paterson & Witt, 2022;Witt et al., 2020). In parts of Africa, the establishment of introduced predators of cochineal elsewhere on the continent poses a potential threat to the management of prickly pear (Paterson & Witt, 2022).
The diversity of cochineal and its specialist natural enemies span North and South America (Portillo, 2009). In the USA, there are a few species in the Southwest, especially in the southern regions of Texas through to California (Badii & Flores, 2001). In Tucson, in southern Arizona, we studied the shared enemies of two local cochineal species, D. opuntiae and Dactylopius confusus (Cockerell). Both species have been recorded as native to the study area (Mann, 1969), but are generally found on different Opuntia species. Dactylopius confusus is most often found on Opuntia engelmanii Salm-Dyck, a native cactus to the southwestern USA. However, we found richer communities of predators and their parasitoids associated with the higher densities of D. opuntiae found on a spineless "Burbank hybrid" of Opuntia ficus-indica (L.) Mill that is common in suburban plantings in the city of Tucson (Anderson & Olsen, 2015). Opuntia ficus-indica is native to central Mexico (Griffith, 2004). Among the natural enemies we observed, we found broad overlap with several key predator specialists described in communities throughout Mexico, and Texas. In all locations, four major predator species predominate: a coccinellid, Hyperapsis trifurcata, an unusual predatory caterpillar, Laetilia coccidivora Comstock, a chamaemyiid fly, Leucopis bellula, and one or more brown lacewing species in the genus Sympherobius (Gilreath & Smith, 1988;Vanegas-Rico et al., 2010).
In Arizona, we found additional cochineal natural enemies that were not, to our knowledge, referenced in the ecological literature: coccinellid beetles that were different in appearance from H. trifurcata, and a parasitoid wasp, Formicencyrtus thoreauini Girault. Here, we characterize two coccinellid beetle phenotypes, eventually, both confirmed as conspecific morphs of an undescribed Hyperaspis sp., with reference to the third common phenotype determined to be H. trifurcata. The two uncharacterized morphs included a common beetle with entirely black elytra of similar size to H. trifurcata, and a rare spotted beetle that appeared in a newly started culture of the black beetle ( Figure 1). Given the broad ecological overlap of our unknown beetle types with H. trifurcata, and the high frequency of color polymorphisms in coccinellids, we initially hypothesized that the black and spotted beetles were a regional color polymorphism of H. trifurcata that had been overlooked. Alternatively, we speculated the black beetles could be Hyperaspis simulans Casey. Gordon (1985) said of H simulans: "The regularly oval form and nearly black, immaculate appearance characterize H. simulans externally, and enable it to be separated from other southwestern species of Hyperaspis." Some specimens labeled as H. simulans in the University of Arizona Insect Collection (UAIC) had been collected from cactus with cochineal and appeared very similar to the black beetles in our culture. We asked the following questions: (1) Are the black and spotted coccinellids color polymorphisms of H. trifurcata, or are they one or two separate species? (2) If the black and spotted beetles are a single separate species, are they two forms of H. simulans? We used morphological (male genitalia) data, molecular phylogenetics, and crossing tests to establish the relationship among the three beetles, and to reveal that the black and spotted beetles are the same species and share mitochondrial COI haplotypes, but are neither H. simulans nor H. trifurcata, but instead appear to be an undescribed species. In additional crosses, we sought to answer one other question: (3) What is the inheritance pattern of the rare spotted phenotype?

T A X O N O M Y C L A S S I F I C A T I O N
Entomology; Life history ecology; Phylogenetics; Taxonomy; Trophic interactions; Urban ecology Second, we show that an unusual-looking flightless ant-mimic parasitoid wasp, Formicencyrtus thoreauini, is a primary parasitoid of D.
opuntiae. Although the type specimen of F. thoreauini is recorded as emerging from Coccus confusus (=Dactylopius confusus) (Girault, 1916) and other museum and host records also record this wasp as being associated with cochineal, it was unclear whether the scale insects or coccinellid beetle larvae were the true hosts. One could easily be misled since beetle larvae often hide within the cochineal wax, perhaps to avoid another ant-mimic encyrtid, Homalotylus cockerelli Timberlake, that has been shown to be a parasitoid of H. trifurcata (Vanegas-Rico et al., 2015). Our initial, incorrect assumption was that F. thoreauini was a beetle parasitoid since we could find no unambiguous records in the literature of parasitism of cochineal, and common knowledge maintains that there are no cochineal parasitoids (e.g., Mendel et al., 2020).

| Beetle collection and culture
We reared H. trifurcata and the black coccinellid that were later shown to be a distinct species in a walk-in rearing room (27°C, 16L:8D). Healthy, mature pads of spineless O. ficus-indica were collected from the field, thoroughly washed and dried, and then infested with crawlers of D. opuntiae in plastic, screened boxes (381 mm × 254 mm × 89 mm). When the cochineal matured, adult beetles were introduced, pupae and adult beetles were harvested from the boxes and held in screened plastic (532 ml) cups containing crumpled laboratory cleaning tissues. The beetles were supplied with water and brewer's yeast mixed with honey.

| Crossing test methods
Experimental beetles were collected and isolated as pupae in 1.2 ml vials plugged with cotton and containing a drop of brewer's yeast and honey mixture. After emergence, the beetles were sexed (male The spotted phenotype of Hyperaspis sp. appeared among the first progeny of a culture started from black individuals collected in the field in Tucson, AZ. Once observed, spotted beetles were segregated into a separate culture. The progeny of these spotted individuals was isolated as pupae, and only spotted parents were used to produce the next generation. For approximately five additional generations, both cultures were observed, and any individuals that differed from the expected phenotype (e.g., "black" in a "spotted" culture or vice versa) were removed. In this way, we strove to increase the homozygosity of color loci prior to performing crosses to investigate the genetic basis for color. We investigated color morph inheritance with a black X spotted cross, a cross of F1 progeny, and a backcross of black males with F1 females. For these crosses, largerscreened containers (241 × 171 × 63 mm) and pieces of cactus pads (at least 80 × 100 mm) were used. Larval offspring were transferred to a fresh pad to complete their development when the cactus piece rotted. For backcrosses, black males drawn from the colony were crossed with isolated virgin F1 females. Most F1 crosses involved isolated individuals, but in a few instances, F1 females which had not been isolated were used for crosses with siblings. For both the F1 × F1 cross and the backcross, results were compiled from single pairs and a single cross of five females and five males. Pupae were collected from each of the crosses, and the number of each phenotype was recorded after adults eclosed. We counted progeny and compared progeny numbers to ratio predictions from Mendelian genetics.

| Morphological analysis of male genitalia
Specimens from the lab cultures were compared to Hyperaspis in the University of Arizona Insect Collection (UAIC) and Gordon (1985) to verify species identification.
Based on external morphology, the lab specimens most closely matched several series of specimens identified as H. simulans. These of Gordon (1985) did not mention cochineal or cactus, and we wondered whether these specimens could have been misidentified.
To further explore species identification, male reproductive structures (from lab-cultured specimens) were examined. Specimens were cleared in cold 10% KOH, rinsed in water, and then passed through progressions of EtOH up to 100% to stop the clearing process. Specimens were disarticulated, slide mounted, and examined using a compound microscope to view genitalia and other morphological characters.

| Molecular phylogenetics and curation of specimens
DNA extractions of beetles were performed with both nondestructive (for later curation of the specimen) and destructive methods. Extractions were performed on Hyperaspis collected in Tucson, AZ, USA, and on laboratory culture specimens, all preserved in 95% EtOH or fresh frozen. Those from ethanol were first rinsed and soaked in water prior to extraction. Initial non-destructive DNA extractions of the beetles involved removing one or two legs, crushing them in a tube with 5 μl of proteinase K (20 mg/ml) and 50 μl of 5%-10% Chelex in water, followed by overnight incubation at 56°C, and a final 8 min incubation at 96°C to inactivate the proteinase K.
This method often did not yield sufficient DNA for amplification, so the abdomens of subsequent specimens were breached and the whole insect was incubated overnight in lysis buffer, followed by standard extraction methods for the Qiagen DNeasy Blood and Tissue kit. Following extraction, the beetles were transferred to 95% ethanol for preservation in the UAIC. Additional destructive extractions were performed using the Qiagen Blood and Tissue Kit. PCR products were quantified, normalized, and sequenced in forward and reverse directions using Sanger sequencing methods at Eton Biosciences or the University of Arizona Genetics Core (UAGC) using an Applied Biosystems 3730 DNA Analyzer (Thermo Fisher Scientific). Chromatograms were assembled into contigs, and initial base calls were made using Phred (Green & Ewing, 2002) & Phrap (Green, 1999) as implemented by the Chromaseq 1.52 (Maddison & Maddison, 2020) module within Mesquite 3.7 (Maddison & Maddison, 2021). Final base calls were made through visual inspection of the contigs. All sequences were submitted to BOLD and GenBank ( Table 3). After trimming, sequences that were at least 500 base pairs long and the sequences obtained specifically for this study were included in the phylogenetic analysis ( Table 3)

| Wasp collection and culture
To distinguish between the hypotheses that F. thoreauini developed as a parasitoid of Hyperaspis or cochineal, field collected wasps were added to boxes with D. opuntiae alone as well as to boxes with D.
opuntiae and H. trifurcata larvae of mixed ages. Some observations of oviposition were made, followed by dissections at various intervals after oviposition. For dissections, cochineal was removed from Opuntia pads, the wax around the insect was removed as much as possible, and the insect was transferred to a drop of saline solution on a microscope slide. The cochineal was dissected with finetip forceps and mounted minuten pins. Images were taken with an Olympus Digital Camera mounted on either a dissecting microscope or a compound microscope.

| Distinguishing the three coccinellid phenotypes
In crossing tests among beetles with the characteristic striped H.
trifurcata phenotype and black and spotted phenotypes, black or spotted beetles paired with H. trifurcata produced no larval progeny (

| Hyperaspis sp.
In the current study, Hyperaspis sp. immature stages differed from The molecular phylogeny of COI sequences confirms the results of crossing tests and morphological analysis (Figure 4), confirming the value of molecular barcoding for uncovering cryptic diversity (Bickford et al., 2007). Hyperaspis sp. consists of two closely related haplotypes, and black and spotted forms were found in both clades.
Although H. trifurcata and Hyperaspis sp. appear embedded in one clade of Hyperaspis, they are not one another's closest relatives ( Figure 4).
It is puzzling that two Hyperaspis species of similar size appear to occupy the same niche on southern Arizona cochineal, in apparent conflict with the competitive exclusion principle (Gause, 1934;Hardin, 1960). It is not uncommon to see both beetles on the same cactus or to even find them next to one another or clustered in the same crevices. We cannot say whether either species occupies additional habitats or attacks alternative prey. However, at least Hyperaspis trifurcata was confirmed to be a specialist prior to being introduced to Israel for biological control (Mendel et al., 2020). More likely, spatial and/or temporal niche partitioning could explain the two beetles' persistence (Amarasekare, 2003). Cactus and cochineal are patchily distributed in both urban and desert landscapes, and our observations suggest that not all predators and parasitoids are found in all patches with cochineal. We have casually observed cochineal undergoing large fluctuations in abundance in a patch over time, reduced by predation and precipitation, and perhaps promoted by dry weather, predator parasitism, and ant protection. As one possible means of coexistence, if one beetle species prevails in interspecific competition within patches, the other could persist by greater dispersal, a type of spatial niche partitioning (Amarasekare, 2003).
At least two other species of Hyperaspis have been observed to feed on cochineal in the USA: Hyperaspis cruenta LeConte (Hunter TA B L E 1 Species limits crosses among "black," "spotted," and H. trifurcata adults. Adults were paired singly or in groups of five females and four to five males in arenas with a portion of a O. ficus-indica pad infested with D. opuntiae. Data presented are the number of crosses in which larvae were produced/total number of crosses performed. No progeny was produced from crosses between H. trifurcata and either "black" or "spotted" phenotypes, but "spotted" and "black" were interfertile. As part of the current study, "black" and "spotted" phenotypes were identified as color morphs of Hyperaspis sp.

F I G U R E 3
Hyperaspis trifurcata larva (a) and Hyperaspis sp. larva (b) and pupa (c) and pupa (d). All images are oriented with the head down. The beetles pupate within the split exuvium of the last larval instar, and the exuvium and the pupa are anchored to the cactus pad by a dried fecal plug that can be seen at the top of (d). The two species' immature stages differ in characteristic color.
Crossing tests designed to investigate the genetic basis of the spotted color morph of Hyperaspis sp. conformed well to the predictions for a single-locus, autosomal-dominant trait ( Table 2). All the progeny of the parental cross were spotted, approximately threefourths of the progeny of the F1 cross were spotted, and half of the progeny of the backcross between F1 females and black males were spotted. Coccinellid color polymorphisms have been investigated in a few common species after pioneering work in Harmonia axyridis (Pallas) by Dobzhansky (Dobzhansky, 1924). Generally, the color pattern is determined by a few genes (Ando & Niimi, 2019;Majerus, 1994Majerus, , 2016. Where a single-locus inheritance pattern is found, the gene could be a transcription factor, or a "supergene," a cluster of genes that are tightly linked, in some cases by inversions, and inherited as if a single locus (Thompson & Jiggins, 2014).
Similarly, the inheritance of color variants of Hyperaspis significans, one with marginal spots and a less common all-black variant, was hypothesized to be due to a single gene, since no intermediate phe- notypes were observed (Dobzhansky, 1941).
Although not sister taxa, the adult Hyperaspis sp. bearing spots were similar in appearance to some color variants seen in H. trifurcata, with less pronounced stripes of cream-colored pigment than in typical H. trifurcata (Figure 1). Indeed, we believed one of our wild-caught specimens to be a spotted morph of Hyperaspis underscoring the value of sequencing for species delineation. It is clear that the rare spotted color morph pattern is a variation on a basic Hyperaspis pattern as hypothesized by Dobhzansky (Dobzhansky, 1941). This ground plan includes five potential spots which can be present or absent and may fuse or vary in shape and exact location according to the species (Dobzhansky, 1941).
Hyperaspis trifurcata most often has all five spots merged into a continuous vitta or stripe extending down from the discal spot, curving around the margin of the elytra (Figure 1), have chemical defenses that are likely to make them distasteful. Eisner et al. (1994) showed that the carmine in the cochineal prey of H. trifurcata was acquired by the beetle and was distasteful to ants. The Old World scale predator Hyperaspis campestris (Herbst) was found to produce "hyperaspine," a novel bitter alkaloid, adding to a list of defensive alkaloids identified in many other coccinellids (Lebrun et al., 2001).

| The parasitoid wasp, F. thoreauini
While we initially hypothesized that F. thoreauini ( Figure 5) was a parasitoid of Hyperaspis immatures, we found no support for this hypothesis, and instead our results indicate that F. thoreauini is a parasitoid of D. opuntiae. We observed females ovipositing into wax containing cochineal (Figure 6), and dissections of cochineal in pure culture of F. thoreauini and D. opuntiae showed eggs and larvae of various stages.
Additionally, we successfully reared two consecutive generations of F.
thoreauini from a pure culture of D. opuntiae in the laboratory. Between TA B L E 2 Exploration of the inheritance pattern of the spotted phenotype in Hyperaspis sp. Before performing the crosses listed here, the black and spotted phenotype beetles were reared in separate cultures for five generations, isolating the pupae and removing the alternative phenotype each generation to try and ensure homozygous parents. "Expected" ratios are those predicted if "spotted" is a single-locus autosomal-dominant trait.  one and four wasps emerged from the mummies that resulted. From these results, we can categorize F. thoreauini as a semi-gregarious wasp, with clutches of one to four or five, and a primary parasitoid of at least D. opuntiae. The type specimen was recorded as being from

Cross-type
Coccus confusus (=D. confusus) (Girault, 1916). Further, when we presented F. thoreauini with both D. opuntiae and Hyperaspis larvae, no beetles became parasitized. In contrast, field collections of infested cactus pads regularly yielded mummified Hyperaspis larvae from which emerged the gregarious beetle parasitoid Homalotylus cockerelli. Prinsloo (1997)  Later instar encyrtid larvae may produce a membrane within which they continue development, and which becomes connected to host tracheae that permit gas exchange with the late/final instar larva and pupa within the membrane (Prinsloo, 1997;Wright, 1986).
In F. thoreauini, a loose membrane was visible surrounding later TA B L E 3 Taxon sampling table and accession numbers for sequences generated specifically for this study. For Hyperaspis sp., we note specimen color morph and clade membership as depicted in Figure 4.

BOLD Accession Color Morph Clade
Hyperaspis instars (Figure 7c), although we cannot be sure of the membrane source, or of host tracheal attachment. Further, each larva appears to pupate in a dry, gas-filled compartment within the cochineal host, likely the remnants of the membrane segregating larva and host hemolymph ( Figure 7d). This adaptation allows a single wasp to successfully complete its development in a mature cochineal, which is not entirely consumed by the larva before pupation (Figure 7d).
When multiple F. thoreauini develop in the same host, each wasp pupates within its own membrane and the entire cochineal is consumed. Smaller-or variable-sized adults have been noted when three or more wasps emerged from a single host. Eclosing adults then chew out of the cochineal mummy, leaving often inconspicuous holes in the cochineal wax ( Figure 7e) of Tucson, AZ, USA, to be hiding in plain sight, within a short walk of the University of Arizona, and within a human community of many active naturalists. These findings underscore that discovery awaits an engaged observer even in apparently well-studied communities.
The geographic range of Hyperaspis sp. is still to be determined.
Formicencyrtus thoreauini was described from specimens collected in New Mexico (Girault, 1916) and is found in Arizona and possibly California (Zuparko, 2015). Neither is described in publications characterizing the communities in Texas (Gilreath & Smith, 1988) nor central Mexico (Vanegas-Rico et al., 2010). How these two community members interact with other natural enemies, cochineal-tending ants, and the many parasitoids and hyperparasitoids in the cochineal community await further exploration.

ACK N OWLED G M ENTS
We had the benefit of the wisdom of several beetle and wasp systematists whom we consulted, including Natalia Vandenberg, Serguei Triapitsyn, John Noyes, Jim Woolley, and Bob Zuparko. We would also like to thank Marco Gebiola for his early interest in the collection of wasps collected off cochineal, and for bringing them to Serguei, who kindly identified them. Nir Netanel and Austin Cruz collected the black Hyperaspis sp. that started our laboratory culture. This work was supported by a US-Israel Binational Science Foundation grant to MSH, Einat Zchori-Fein, and Moshe Inbar (#2016040) and the study benefitted from our close collaboration on cochineal and Hyperaspis.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data (sequence information as well as sample metadata) can be accessed at BOLD and GenBank (see Table 3 for accession numbers).